Activation of encrypted tissue factor during thrombus formation in vivo

作为一种宿主防御机制，凝血级联反应的启动需要严格的调控 无论是在时间上还是在地点上。主要死因，包括中风、心脏病发作和肺病 栓塞症，每个都与病理性血栓形成有关。目前的凝血教学是从 对这一复杂系统的分离成分进行单独研究的体外研究。我的长期生活 目的是阐明体内启动凝血的机制，并确定这一关键步骤是如何 受监管的。最近发展起来的成像技术使研究蛋白质相互作用成为可能 在活体动物体内实时形成血栓。组织因子(TF)是启动血液凝固所必需的， 凝血酶形成和纤维蛋白生成。人们一直认为，组织因子不会受到 血液循环，只有血管损伤才会暴露。然而，Tf已经在循环中被确认 微粒，以及白细胞和内皮细胞上的微粒。最近的研究导致了这部小说 认为Tf的氧化导致二硫键的形成，起到分子开关的作用 潜伏的转铁蛋白转化为促凝剂。我们假设在循环中存在一种加密形式的TF 在体内血栓形成和纤维蛋白生成过程中被激活。这将是一种新的机制 血管紧张素转运蛋白参与血栓形成。我们基于以下观察结果提出这一假设：1)在体内 循环转铁蛋白在血栓生长中积聚的证据，2)循环转铁蛋白之间的差异 用抗原和功能循环抗原测量的浓度远远超过 启动凝血和3)破坏组织因子中的Cys186-Cys209二硫键降低其 促凝血活性，这是可逆的，通过形成同样的二硫键。基于这些 观察，我们将使用活体内表征体内活性和加密的组织因子亚型 显微镜和异构体特异性抗体。我们将鉴定已知抗体的异构体特异性。 并利用重组噬菌体展示技术开发了新型抗体。这些抗体将 在激光诱导的活体小鼠血栓形成过程中，允许对转铁蛋白亚型进行可视化。此外， 在单个感兴趣的组织中，包括单核细胞和内皮细胞，缺乏转铁蛋白的小鼠模型将 被用来研究这些组织在体内TF介导的血栓形成中的作用。

As a mechanism of host defense, the initiation of the blood coagulation cascade requires tight regulation both in its timing and location. Major causes of death, including stroke, heart attack and pulmonary embolism, are each linked to pathologic thrombosis. Current teaching of coagulation has evolved from in vitro studies in which isolated components of this complex system are studied individually. My long-term goal is to elucidate the mechanism initiating coagulation in vivo and to determine how this critical step is regulated. Recently developed imaging technologies allow for the study of protein interactions at the site of thrombus formation in a living animal in real time. Tissue factor (TF) is required to initiate bloodcoagulation, thrombin formation and fibrin generation. It has been dogma that tissue factor is shielded from the circulation and is only exposed by vascular injury. However, TF has been identified on circulating microparticles, as well as on leukocytes and endothelial cells. Recent studies have led to the novel proposal that oxidation of TF leads to disulfide bond formation, acting as a molecular switch that changes latent TF to a procoagulant form. We hypothesize that an encrypted form of TF exists within the circulation that is activated during thrombus formation and fibrin generation in vivo. This would be a novel mechanism of TF involvement in thrombosis. We base this hypothesis on the following observations: 1) in vivo evidence of circulating TF accumulating in a growing thrombus, 2) a discrepancy between circulating TF concentration as measured by antigen and function - circulating antigen far exceeds the amount required to initiate coagulation and 3) disruption of the Cys186-Cys209 disulfide bond in tissue factor decreases its procoagulant activity, and this is reversible by forming this same disulfide bond. Based on these observations, we will characterize active and encrypted tissue factor isoforms in vivo using intravital microscopy and isoform-specific antibodies. We will characterize known antibodies for isoform specificity and have developed novel antibodies using recombinant phage display technology. These antibodies will allow visualization of TF isoforms during laser-induced thrombus formation in a living mouse. Furthermore, mouse models deficient in TF in individual tissues of interest, including monocytes and endothelial cells, will be employed to investigate the role of these tissues in TF-mediated thrombus formation in vivo.